Prescription opioid narcotics, such as morphine, oxycodone, and fentanyl, produce analgesia and side effects through activation of the mu opioid receptor (MOR), a G protein coupled receptor (GPCR). Our long-standing goal is to understand how MOR signals to produce distinct biological effects and to ultimately inform the development of therapeutics that will take advantage of good receptor signaling (pain relief) and avoid bad receptor signaling (tolerance, dependence, constipation and other side effects). It has become increasingly evident that different drug structures can elicit different receptor signaling cascade at a single receptor, likely by changing the affinities for association with intracellular binding partners. Further, the intracellular binding partner profile differs between neuronal populations. Therefore, the nature of a drug response can be determined not only by the chemical properties of the drug, but also by the complement of signaling proteins found in residence with the receptor; making it critical to study receptor signaling in physiologically relevant systems. One particular intracellular protein that influences MOR function is ?arrestin2. betaArrestin2 is a scaffolding protein that can act as desensitizing element or as a signal transduction facilitator. Our studies have shown that morphine-induced analgesia is enhanced while tolerance is attenuated in mice lacking betaarrestin2, which implicates betaarrestin2 as a desensitizing factor in pain regulating brain regions. Our collective body of work shows that the severity of certain side effects, including physical dependence and constipation, are significantly reduced in mice lacking betaarrestin2 suggesting that in some organ systems and brain regions, betaarrestin2 facilitates MOR signaling. Since receptor responsiveness to a drug in vivo is ultimately dependent upon the cellular environment that encompasses the receptor, we hypothesize that betaarrestin2 dampens morphine responsiveness in analgesia pathways while it mediates morphine-associated side effects such as physical dependence and constipation. To this end, we propose to elucidate the mechanisms by which betaarrestins regulate MOR in brain regions and tissues that mediate morphine-induced antinociception and tolerance (brainstem), physical dependence (striatum) and constipation (colon). We will utilize new MOR agonists that are functionally selective for activating G protein signaling pathways (we hypothesize this will promote antinociception) and against recruiting betaarrestin2 (we hypothesize that recruiting ?arrestin2 leads to tolerance, dependence and constipation). Published and preliminary evidence suggests that the G protein biased agonists promote antinociception with fewer side effects. We will use these tools to gain a greater understanding of MOR regulation in the endogenous setting as it pertains to in vivo physiologies. These studies should provide guidance for developing therapeutics that preferentially enhance desired effects such as improving pain therapy while preventing adverse reactions.